Day1 Dissection: • 3-5 days old flies are dissected in ice-cold Schneider’s Insect Medium.
{"action": "examine", "output": "", "time": [""]}
• Dissected tissues are fixed in 2% PFA at 25 ̊ C for 55 min. #####%%%%% output "graded EtOH series" are used in line6
{"action": "reset", "output": "graded EtOH series", "time": ["55 min"]}
• Fixed tissues are washed with 0.5% PBT, 3x, 10 min each Dehydration: • Dehydrate the fixed tissues with graded EtOH series: 30%, 50%, 70%, 100%, 100%, 100%, 10 min each.
{"action": "wash", "output": "", "reagent": ["0.5% PBT"], "time": ["10 min"]}
• Dehydrated tissues are stored at 4 ̊C with 100% EtOH overnight on a rocker. #####%%%%% output "graded EtOH series" are used in line10
{"action": "use", "output": "graded EtOH series", "device": ["a rocker"], "temperature": ["4 \u030aC"]}
Day2 Permeation: • 70%, 50%, 30%, 10 min eachDehydrated tissues are rehydrated with graded EtOH series: .
{"action": "rinse", "output": "", "time": ["10 min"], "reagent": ["graded EtOH series"]}
• Rehydrated tissues are incubated at 4 ̊C 5% acetic acid for 5 min \(do this in the cold room ).
{"action": "operate", "output": "", "reagent": ["5% acetic acid"], "device": ["cold room"], "temperature": ["4 \u030aC"], "time": ["5 min"]}
• Fix tissues with 2% PFA at 25 ̊C for 55 min. #####%%%%% output "tissue" are used in line16
{"action": "correct", "output": "tissue", "concentration": ["2% PFA"], "temperature": ["25 \u030aC"], "time": ["55 min"]}
• Wash tissue with 0.5% PBT, 3x, 10 min each.
{"action": "wash", "output": "", "reagent": ["0.5% PBT"], "time": ["10 min"]}
• Wash with 4 ̊C 1xPBS for 3x, 5 min each. #####%%%%% output "pre-hybr soln" are used in line20
{"action": "wash", "output": "pre-hybr soln", "temperature": ["4 ̊C"], "time": ["5 min each"], "reagent": ["PBS"], "force": [""]}
Pre-Hybridization: • Prepare fresh pre-hybr soln with formamide • Incubate tissues with pre-hybr soln at 50 ̊C for 2 hrs.
{"action": "prepare", "output": "", "reagent": ["pre-hybr soln"]}
Hybridization: • Incubate FISH probes in 90 ̊C for 3 min, then snap cold the FISH probes on ice for 10 min.
{"action": "grab", "output": "", "device": [""]}
• Prepare ~48μL #####%%%%% output "the pre-hybr soln" are used in line26
{"action": "prepare", "output": "the pre-hybr soln", "volume": ["~48\u03bcL"]}
Day3 Washing: • Warm up the pre-hybr soln to 37 ̊C. #####%%%%% output "the hybridization reaction" are used in line28
{"action": "soak", "output": "the hybridization reaction", "reagent": ["the pre-hybr soln"], "temperature": ["37 \u030aC"]}
• Add 200μL 37 ̊C pre-hybr soln directly to the hybridization reaction, .
{"action": "add", "output": "", "reagent": ["the hybridization reaction"]}
incubate for 10 min #####%%%%% output "pre-hybr soln" are used in line32
{"action": "incubate", "output": "pre-hybr soln", "time": ["10 min"], "temperature": [""]}
• Wash with pre-hybr soln, 1x, 37 ̊C, 10 min. #####%%%%% output "pre-hybr soln" are used in line34
{"action": "wash", "output": "pre-hybr soln", "reagent": ["pre-hybr soln"]}
• Wash with pre-hybr soln, 1x, 25 ̊C, 10 min. #####%%%%% output "30% formamide washing soln" are used in line36
{"action": "wash", "output": "30% formamide washing soln", "reagent": ["pre-hybr soln"]}
• Wash with 30% formamide washing soln, 2x, 25 ̊C, 30 min each . #####%%%%% output "2xSSC washing soln" are used in line38
{"action": "wash", "output": "2xSSC washing soln", "reagent": ["30% formamide washing soln"]}
• Wash with 2xSSC washing soln, 3x, at 25 ̊C, 10 min each • Wash with 1xPBS, 1x, at 25 ̊C, 10 min • Fix tissues at 2% PFA at 25 ̊C for 55 min. #####%%%%% output "each" are used in line40
{"action": "wash", "output": "each", "reagent": ["2xSSC washing soln"]}
• Wash with 0.5% PBT, 3x, at 25 ̊C, 10 min each.
{"action": "wash", "output": "", "temperature": ["25 \u030aC"], "time": ["10 min each"], "force": [""], "reagent": ["each"]}
• Dehydrate the tissues with graded EtOH series: 30%, 50%, 75%, 100%, 100%, 100%, 10 min each. #####%%%%% output "BB-SIM" are used in line44
{"action": "dehydrate", "output": "BB-SIM", "concentration": ["30%, 50%, 75%, 100%, 100%, 100%, 10 min each"]}
• For BB-SIM, hold tissues in xylene until imaging.
{"action": "hold", "output": "", "reagent": ["BB-SIM"]}